Filtern
Erscheinungsjahr
Dokumenttyp
- Dissertation (156)
Volltext vorhanden
- ja (156)
Gehört zur Bibliographie
- nein (156)
Schlagworte
- Paracoccus denitrificans (7)
- Cytochromoxidase (6)
- Ubihydrochinon-Cytochrom-c-Reductase (6)
- ABC-Transporter (4)
- Membranproteine (4)
- Elektronentransfer (3)
- Protonentransfer (3)
- Apoptosis (2)
- Atmungskette (2)
- Biochemie (2)
Institut
- Biochemie und Chemie (131)
- Biowissenschaften (11)
- Pharmazie (8)
- Biochemie, Chemie und Pharmazie (3)
- Georg-Speyer-Haus (3)
- Medizin (2)
- MPI für Biophysik (1)
- Physik (1)
Untersuchungen zur Bedeutung von Superoxid-Dismutasen für die Alterung von Podospora anserina
(2012)
Im Rahmen dieser vorliegenden Doktorarbeit sollte die Bedeutung von Superoxid-Dismutasen für das Resistenzverhalten und den Alterungsprozess bei P. anserina untersucht werden. Folgende Befunde aus den Analysen konnten erhalten werden:
1. Lokalisationsstudien der drei PaSods: Aus den biochemischen und fluoreszenzmikroskopischen Untersuchungen der drei verschiedenen PaSODs geht hervor, dass PaSOD1, eine Cu/ZnSOD, überwiegend im Cytosol und zu einem geringen Anteil im mitochondrialen Intermembranraum lokalisiert ist. Eine der beiden MnSODs, PaSOD2, wird vermutlich zur Abwehr von exogenem Superoxid sekretiert. Bei PaSOD3 handelt es sich um eine mitochondriale MnSOD.
2. Generierung von verschiedenen PaSod-Mutanten: Im Rahmen dieser Arbeit wurden von jeder PaSod mindestens drei unabhängige Überexpressionsstämme, ein GFP-Stamm- und ein Deletionsstamm hergestellt. Weiterhin wurden alle möglichen Doppel-Deletionsstämme und die Dreifach-Deletionsmutante erzeugt. Alle Stämme wurden auf DNA-Ebene verifiziert, zusätzlich wurde die Proteinmenge bzw. –Aktivität überprüft.
3. Einfluss der PaSODs auf die ROS-Toleranz: Die Analysen der ROS-Resistenzen haben gezeigt, dass PaSODs eine wichtige Rolle in der Entgiftung von Superoxiden spielt. So ließ sich bei den Deletionsstämmen der PaSods eine gesteigerte Sensitivität gegenüber Paraquat feststellen. Eine Aufsummierung der Sensitivität gegenüber Paraquat ist bei der PaSod-Tripelmutante (ΔPaSod1/2/3) zu erkennen.
Überraschenderweise kann durch die gesteigerten Mengen an aktiver PaSOD in den Überexpressionsstämmen (PaSod1-3_OEx) keine verbesserte Resistenz gegenüber Paraquat erzielt werden. Darüber hinaus führt die Überexpression des Gens für die mitochondriale SOD, PaSOD3, zu massiven negativen Effekten.
4. Einfluss auf die Lebensspanne: Durch eine fehlende Entgiftung von Superoxid in den PaSod-Deletionsmutanten ist eine Verminderung der Lebensspanne nicht festzustellen. Bei PaSod-Mutantenstämme, die eine erhöhte PaSOD-Aktivität und damit eine gesteigerte Abbaurate des Superoxids aufweisen, kann bei den PaSod1- und PaSod2-Überexpressionsstämmen keine verbesserte Lebensspanne unter den gewählten Standardbedingungen erzielt werden. Vielmehr noch ist die Lebensspanne der PaSod3-Überexpressionsstämme stark reduziert.
5. Einfluss der PaSod-Modulation auf andere Komponenten des ROS-Abbausystems: Die PaSOD-Aktivitäten scheinen miteinander co-reguliert zu werden. Des Weiteren scheint es ein Zusammenhang zwischen den beiden sekretierten Enzymen PaSOD2 und PaCATB zu geben. Deutlich wird auch, dass die Modulation der Superoxid-Dismutasen eine weitreichende Auswirkung auf andere Schutzsysteme hat. Beispielweise konnte gezeigt werden, dass Komponenten des mitochondrialen ROS-Schutzsystems und der Protein-Qualitätskontrolle in den PaSod3-Überexpressionsstämmen verändert sind.
Zusammenfassend lassen die Analysen der PaSod-modulierten Stämme den Schluss zu, dass die Superoxid-Dismutase in P. anserina ein wichtiges Enzym zum Abbau des schädlichen Superoxids darstellt, welches aber nur eine untergeordnete Rolle bei der Kontrolle der Lebensspanne unter den gewählten Wachstumsbedingungen im Labor ausübt. Des Weiteren haben die Analysen gezeigt, dass es durch die Modulation der PaSod-Gene zu weitreichenden Änderungen, die das ROS-Schutzsystem (PaSOD, PaCATB und PaPRX1) sowie die Protein-Qualitätskontrolle (PaHSP60, PaLON und PaCLPP) betreffen, kommt. Welche Auswirkung dabei diese Veränderungen in Bezug auf die Lebensspanne hat, kann nur schwer abgeschätzt werden und muss mit weiteren Untersuchungen geklärt werden.
The technique of site-specific fluorescence labelling with Tetramethylrhodaminemaleimide (TMRM) in combination with two electrode voltage-clamp technique (TEVC), an approach that has been named voltage clamp fluorometry (VCF), has been used in this work to study the Na,K-ATPase. The TMRM dye has the ability to attach covalently to cysteine residues and it responds to changes in the hydrophobicity of its local environment. We exploited this property using a construct of the Na-pump in which the native, extracellularly accessible cysteines were removed and cysteine residues were introduced by site-directed mutagenesis in specific positions of the Na-pump. In this way it was possible to detect site-specific conformational rearrangements of the Na-pump in a time-resolved fashion within a native membrane environment. In particular this technique allows to resolve reactions with low electrogenicity that cannot be satisfactorily analyzed with purely electrophysiological techniques and to identify the conformations of the enzyme under specific ionic composition of the measuring buffers. We used VCF to study the influence that several cations like Na+, K+, NMG+, TEA+ and BTEA+ exert on the distribution of the Na,K-ATPase between several enzymatic intermediates and on some of the reactions related to cation transport. To this end we utilized the mutants N790C in the loop M5-M6 and the mutant E307C, T309C, L311C and E312C in the loop M3-M4. From the correspondence of the fluorescence changes with the activation and inhibition of pumping current, by K+ and ouabain respectively, and from the fact that in Na+/Na+ exchange conditions the voltage distribution of charge movement and fluorescence changes evoked by voltage jumps are in reasonable agreement we conclude that through the fluorescence signals measured from these mutants, we can indeed monitor conformational changes linked to transport activity of the enzyme. For the mutants N790 and L311, it was found that the Na+ dependence of the amplitude and kinetics of the fluorescence signal associated with the E1P-E2P transition is in agreement with the prediction of an access channel model describing the regulation of the access of extracellular Na+ to its binding site. In particular for the mutants E307 and T309 it was found that in Na+/Na+ exchange conditions, the conformational change tracked by the fluorescence was much slower than the charge relaxation at hyperpolarized potentials while the kinetics was very similar at depolarized potentials. This implies that at hyperpolarized potentials the conformational change connected to the E1P-E2P transition does not give a large contribution to the electrogenicity of the process which is also consistent with the access channel model. On the mutant N790C it was found that the external pH does not seem to have any effect on the E1P-E2P equilibrium even if it seems to modulate the fluorescence quantum yield of the dye. Fluorescence quenching experiments with iodide and D2O indicate that at hyperpolarized potentials the local environment of the mutant N790C, experiences a small change in the accessibility to water without major changes in the local electrostatic field ...
The Na+/proline transporter of E. Coli (PutP) is responsible for the uptake of proline which is subsequently used not only as a carbon and nitrogen source and a constituent of proteins but also as a particularly effective osmoprotectant. However, for a long time there was little known about the single steps in the reaction cycle of this transporter and only few details about its structure-function relationship are available. Aim of the present work was to achieve a deeper understanding about the kinetic properties of the Na+/proline transporter and to get insights into the structure-function relationship of the substrate binding. To answer these questions different techniques were used. By using the novel SSM technique combining the preparation of PutP proteoliposomes it was possible to demonstrate for the first time the electrogenic substrate binding to PutP transporter. Due to rapid solution exchange measurements on the SSM it was additionally possible to obtain time resolved information about the kinetic details of the cytoplasmic substrate binding sites which were not available by previous steady state and equilibrium binding measurements. Pre-steady-state charge translocation was observed after rapid addition of one or both of the cosubstrates Na+ and/or proline to the PutP-WT proteoliposomes adsorbed on the SSM. Thereby it was possible to link the observed electrical signals with the binding activity of PutP. The observed Na+ and/or proline induced charge displacement were assigned to an electrogenic Na+ and/or proline binding process at the cytoplasmic face of the enzyme with a rate constant of k > 50 s-1 proceeding the rate limiting step of the reaction cycle. Furthermore, based on the kinetic analysis of the electrical signals obtained from the measurements of PutP on SSM, the following characteristics of the substrates binding in PutP were deduced: (1) both Na+ and proline can bind individually to the transporter. Under physiological conditions, an ordered binding mechanism prevails; while at sufficiently high concentrations, each substrate can bind in the absence of the other; (2) substrate binding is electrogenic not only for Na+, but also for the uncharged cosubstrate proline. The charge displacement associated with Na+ binding and proline binding is of comparable size and independent of the presence of the respective cosubstrate. In addition, it was concluded that Na+ accesses its binding site through a high-field access channel resulting in a charge translocation, whereas the binding of the electroneutral proline induces a conformation alteration involving the displacement of charged amino acid residue(s) of the protein; (3) Na+ and proline binding sites interact cooperatively with each other by increasing the affinity and/or the speed of binding of the respective cosubstrate; (4) proline binding proceeds in a two step process: low affinity (~ 0.9 mM) electroneutral substrate binding followed by a nearly irreversible electrogenic conformational transition; (5) membrane impermeable PCMBS inhibits both Na+ and proline binding to the inside-out orientated PutP transporter, indicating that rather than selectively blocking a specific binding site, PCMBS probably locks the enzyme in an inactive state. The possible targets for this SH-reagent are cysteines 281 and 344 located close to the cytoplasmic surface of the protein. Beyond it, transient electrical currents of PutP were also observed on the BLM after rapid addition of proline in the presence of Na+. This was possible by combining the conventional BLM technique with high-speed flash-photolysis of caged-proline. Indeed the signals on the BLM indicate the detection of a different underlying reaction process in comparison to the data achieved by the SSM technique. This has paved the way for supplemental information about the reaction cycle since it was possible to assign the flash-photolysis BLM signals to the proline binding step followed by the internalization of Na+ and proline into the liposome. Thereby it was found, that the presence of Na+ is indispensable and the time constant for the process is ~ 63 ms. Moreover, structure-function information about the Na+ and proline binding sites of PutP was obtained by investigating the functionally important amino acid residues Asp55, Gly63 and Asp187 with site-directed mutagenesis and the combined SSM technique. One finding is that the mutated proteins PutP-D55C and PutP-G63C showed no activity on the SSM. Therefore, it can be assumed that either both Asp55 and Gly63 are crucial for the structure of PutP protein, or they are located at or close to the Na+ and proline binding sites. Furthermore, the results obtained from PutP-D187N and PutP-D187C mutants on SSM suggest that Asp187 of PutP is likely to be involved in the Na+ binding at the cytoplasmic side of the backward running carrier. Taken together the results of the present work have substantially broadened the known picture of the Na+/proline transporter PutP thereby several steps of the reaction cycle were elucidated, and moreover, valuable insights into the structure-function relationship of the transporter have become available.
This work presents a biochemical, functional and structural characterization of Aquifex aeolicus F1FO ATP synthase obtained using both a native form (AAF1FO) and a heterologous form (EAF1FO) of this enzyme.
F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane and therefore play a key cellular function. Because of their central role in supporting life, F1FO ATP synthases are ubiquitous and have been remarkably conserved throughout evolution. For their biological importance, F1FO ATP synthases have been extensively studied for many decades and many of them were characterized from both a functional and a structural standpoint. However, important properties of ATP synthases – specifically properties pertaining to their membrane embedded subunits – have yet to be determined and no structures are available to date for the intact enzyme complex. Therefore, F1FO ATP synthases are still a major focus of research worldwide. Our research group had previously reported an initial characterization of AAF1FO and had indicated that this enzyme presents unique features, i.e. a bent central stalk and a putatively heterodimeric peripheral stalk. Based on such a characterization, this enzyme revealed promising for structural and functional studies on ATP synthases and became the focus of this doctoral thesis. Two different lines of research were followed in this work.
First, the characterization of AAF1FO was extended by bioinformatic, biochemical and enzymatic analyses. The work on AAF1FO led to the identification of a new detergent that maintains a higher homogeneity and integrity of the complex, namely the detergent trans-4-(trans-4’-propylcyclohexyl)cyclohexyl-α-D-maltoside (α-PCC). The characterization of AAF1FO in this new detergent showed that AAF1FO is a proton-dependent, not a sodium ion-dependent ATP synthase and that its ATP hydrolysis mechanism needs to be triggered and activated by high temperatures, possibly inducing a conformational switch in subunit γ. Moreover, this approach suggested that AAF1FO may present unusual features in its membrane subunits, i.e. short N-terminal segments in subunits a and c with implications for the membrane insertion mechanism of these subunits.
Investigating on these unique features of A. aeolicus F1FO ATP synthase could not be done using A. aeolicus cells, because these require a harsh and dangerous environment for growth and they are inaccessible to genetic manipulations. Therefore, a second approach was pursued, in which an expression system was created to produce the enzyme in the heterologous host E. coli. This second approach was experimentally challenging, because A. aeolicus F1FO ATP synthase is a 500-kDa multimeric membrane enzyme with a complicated and still not entirely determined stoichiometry and because its encoding genes are scattered throughout A. aeolicus genome, rather than being organized in one single operon. However, an artificial operon suitable for expression was created in this work and led to the successful production of an active and fully assembled form of Aquifex aeolicus F1FO ATP synthase. Such artificial operon was created using a stepwise approach, in which we expressed and studied first individual subunits, then subcomplexes, and finally the entire F1FO ATP synthase complex. We confirmed experimentally that subunits b1 and b2 form a heterodimeric subcomplex in the E. coli membranes, which is a unique case among ATP synthases of non-photosynthetic organisms. Moreover, we determined that the b1b2 subcomplex is sufficient to recruit the soluble F1 subcomplex to the membranes, without requiring the presence of the other membrane subunits a and c. The latter subunits can be produced in our expression system only when the whole ATP synthase is expressed, but not in isolation nor in the context of smaller FO subcomplexes. These observations led us to propose a novel mechanism for the assembly of ATP synthases, in which first the F1 subcomplex attaches to the membrane via subunit b1b2, and then cring and subunits a assemble to complete the FO subcomplex. Furthermore, we could purify the heterologous ATP synthase (EAF1FO) to homogeneity by chromatography and electro-elution. Enzymatic assays showed that the purified form of EAF1FO is as active as AAF1FO. Peptide mass fingerprinting showed that EAF1FO is composed of the same subunits as AAF1FO and all soluble and membrane subunits could be identified. Finally, single-particle electron microscopy analysis revealed that the structure of EAF1FO is identical to that of AAF1FO. Therefore, the EAF1FO expression system serves as a reliable platform for investigating on properties of AAF1FO.
Specifically, in this work, EAF1FO was used to study the membrane insertion mechanism of rotary subunit c. Subunits c possess different lengths and levels of hydrophobicity across species and by analyzing their N-terminal variability, four phylogenetic groups of subunits c were distinguished (groups 1 to 4). As a member of group 2, the subunit c from A. aeolicus F1FO ATP synthase is characterized by an N-terminal segment that functions as a signal peptide with SRP recognition features, a unique case for bacterial F1FO ATP synthases. By accurately designing mutants of EAF1FO, we determined that such a signal peptide is strictly necessary for membrane insertion of subunit c and we concluded that A. aeolicus subunit c inserts into E. coli membranes using a different pathway than E. coli subunit c. Such a property may be common to other ATP synthases from extremophilic organisms, which all cluster in the same phylogenetic group.
In conclusion, the successful production of the fully assembled and active F1FO ATP synthase from A. aeolicus in E. coli reported in this work provides a novel genetic system to study A. aeolicus F1FO ATP synthase. To a broader extent, it will also serve in the future as a solid reference for designing strategies aimed at producing large multi-subunit complexes with complicated stoichiometry.
RNA modifications are widespread in the RNA world. Nevertheless, their functions remain enigmatic. Recent analysis in tRNAs, mRNAs and rRNAs have revealed that apart from enriching their topological potential, these chemical modifications provide an added significant regulatory level to gene expression...
Heme-copper oxidases (HCOs) are the terminal enzymes of the aerobic respiratory chain in the inner mitochondrial membrane or the plasma membrane in many prokaryotes. These multi-subunit membrane protein complexes catalyze the reduction of oxygen to water, coupling this exothermic reaction to the establishment of an electrochemical proton gradient across the membrane in which they are embedded. The energy stored in the electrochemical proton gradient is used e.g. by the FOF1-ATP synthase to generate ATP from ADP and inorganic phosphate. The superfamily of HCOs is phylogenetically classified into three major families: A, B and C. The A-family HCOs, represented by the well-studied aa3-type cytochrome c oxidases (aa3-CcOs), are found in mitochondria and many bacteria. The B-family of HCOs contains a number of bacterial and archaeal oxidases. The C-family comprises only the cbb3-type cytochrome c oxidase (cbb3-CcO) and is most distantly related to the mitochondrial respiratory oxidases.
1. Fab co-complexes of proton pumping NADH:ubiquinone oxidoreductase (complex I) Fab fragments suitable for co-crystallization with complex I were generated using an immobilized papainbased protocol. The binding of the antibody fragments to complex I was verified using Surface Plasmon Resonance and size exclusion chromatography. The binding constants of the antibodies and their respective Fab fragments were found to be in the nanomolar range. This work presents the first report on successful crystallization of complex I (proton pumping NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica with proteolytic Fab fragments. The quality of the crystals was significantly improved when compared to the initial experiments and the best crystals diffracted X-rays to a resolution of ~7 Å. The activity of complex I remained uninfluenced by antibody fragment binding. The initial diffraction data suggest that the complex I/Fab co-complex crystals represent a space group different to the one observed for the native protein. Ongoing experiments are aimed at further enhancements of the diffraction quality of the crystals. Providing a different space group the CI/Fab co-complexes may become a very useful approach for structure determination of the enzyme. Moreover, the bound Fab offers an additional possibility to generate phase information. The antibody-mediated crystallization represents a valuable tool in structural characterization of the NADH:oxidoreductase subcomplexes or even single subunits. 2. UDP-glucose pyrophosphorylase UDP-glucose pyrophosphorylase from Yarrowia lipolytica displays affinity towards Ni2+ NTA and was first detected in a contaminated sample of complex I. Following, separation from complex I, Ugp1p was purified using anion exchange chromatography. Sequence similarity studies revealed high identity to other known pyrophosphorylases. As indicated by laser-based mass spectrometry method (LILBID) Ugp1p from Y. lipolytica builds octamers similarly to the enzyme from Saccharomyces cerevisiae. The initial crystals grew as thin needles favorably in sitting drop setups. The size of the crystals was increased by employment of a micro batch technique. The improved crystals diffracted X-rays to a resolution of 3.2 Å at the synchrotron beamline. Structural characterization is under way using a molecular replacement approach based on the published structure of baker’s yeast UGPase.
Plants absorb sunlight via photosynthetic pigments and convert light energy intochemical energy in the process of photosynthesis. These pigments are mainly bound to antenna protein complexes that funnel the excitation energy to the photosynthetic reaction centres. The peripheral antenna of plant photosystem II (PSII) consists of the major light-harvesting complex of PSII (LHC-II) and the minor LHCs CP29, CP26 and CP24. Light intensity can change frequently and plants need to adapt to high-light conditions in order to avoid photodamage. When more photons are absorbed than can be utilised by the photosynthetic machinery, excessive excitation energy is dissipated as heat by short-term adaptation processes collectively known as non-photochemical quenching (NPQ). A decrease in PSII antenna chlorophyll (Chl) fluorescence yield and a reduction in the average Chl fluorescence lifetime are associated with NPQ. The main component of NPQ is the so-called energy-dependent quenching (qE), and it is triggered by the rapid drop in thylakoid lumenal pH resulting from the plant’s photosynthetic activity. This process is thought to take place at the PSII antenna complexes, which therefore not only capture and transfer light energy but are also involved in balancing the energy flow. The decrease in lumenal pH acivates the enzyme violaxanthin de-epoxidase (VDE), which converts the xanthophyll violaxanthin (Vio) into zeaxanthin (Zea) in the xanthophyll cycle. In addition, the PSII subunit PsbS was discovered to be essential for qE by screening qE-deficient Arabidopsis thaliana mutants. This membrane protein is considered a member of the LHC superfamily, which also includes LHC-II and the minor LHCs. Previous studies on PsbS isolated either from native source or refolded in vitro have produced inconsistent results on its pigment binding capacity. Interestingly, a pH-dependent change in the quaternary structure of PsbS under high light conditions has been reported. This observed dimer-tomonomer transition very likely follows the protonation of lumenal glutamates upon the drop in pH and is accompanied by a change in PSII supercomplex localisation. PsbS dimers are preferentially found in association with the PSII core, whereas PsbS monomers co-localise with LHC-II.Despite the identification of !pH, Zea and PsbS as key players in qE, both the nature of the quencher(s) as well as the underlying molecular mechanism leading to excess energy dissipation still remain unknown. Several models have been put forward to explain the reversible switch in the antenna from an energy-transmitting to a quenched state. Proposals include a simple pigment exchange of Vio for Zea, and aggregation or an internal conformational change of LHC-II. Charge transfer (CT)quenching in the minor LHCs or quenching by carotenoid dark state (Car S1)-Chl interactions have also been suggested. However, none of these qE models has so far been capable of accommodating all the physiological observations and available experimental data. Most importantly, the function of PsbS remains an enigma. A recent qE model suggested that monomerisation of PsbS enables the protein to transiently bind a carotenoid and form a quenching unit with a Chl of a PSII LHC. In view of the various proposed qE mechanisms, this thesis aimed at understanding the interplay of the different qE components and the contribution of the PSII subunits LHC-II, the minor LHCs and PsbS to qE. The initial approach was to investigate the properties of the PSII subunits in the most simple in vitro model system, namely in detergent solution. For this purpose, LHC-II was isolated either from native source or refolded from recombinantly produced protein. Investigation of the minor LHCs and PsbS required heterologous expression and refolding. In addition, experiments were performed on aggregated LHC-II. Aggregates of LHC-II have been used as a popular model system for qE because they exhibit highly quenched Chl fluorescence. At the final stage of this doctoral work, a more sophisticated model system to approximate the thylakoid membrane was developed by reconstitution of the PSII subunits LHC-II and PsbS into liposomes. This system not only allowed for investigation of these membrane proteins in their native environment, but also for mimicking the xanthophyll cycle by distribution of Zea within the membrane as well as !pH by outside buffer exchange. The role of Zea in qE was first investigated with detergent solubilised antenna proteins. The requirement of this xanthophyll for qE is well-known, but the specific contribution to the molecular quenching mechansim is unclear. Previous work had shown that replacement of Vio for Zea in LHC-II was not sufficient to induce Chl fluorescence quenching in Zea-LHC-II, as suggested by the so-called molecular gearshift mechanism. However, by means of selective two-photon excitation spectroscopy, an increase in electronic interactions between Car S1 and Chls was observed for LHC-II upon lowering the pH of the detergent buffer. Electronic Car S1-Chl coupling became even stronger when Zea-LHC-II was probed. The extent of Car S1-Chl coupling correlated directly with the extent of Chl fluorescence quenching, in a similar way as observed previously in live plants under high-light conditions. However, very similar results were obtained with LHC-II aggregates. This implied that the increase in electronic interactions and fluorescence quenching was independent of Zea and low pH. Further experiments on aggregates of LHC-II Chl mutants indicated that the targeted pigments were also not essential for the observed effects. It is proposed that the same molecular mechanism causes an increase in electronic Car S1-Chl interactions and Chl fluorescence quenching in Zea-LHC-II at low pH as well as in aggregated LHC-II. Most likely, surface exposed pigments form random quenching centres in both cases. On the other hand, it was possible that Zea could act as a direct quencher of excess excitation energy in the minor LHCs. However, enrichment of refolded CP29, CP26 and CP24 with Zea did not lead to a change in the Chl excited state lifetime. Formation of a carotenoid radical cation, previously implied in CT quenching, was also not observed, although artificial generation of such a radical cation was principally possible as shown for CP29. During the course of this work, a study reporting the formation of Zea radical cations in minor LHCs was published. Therefore, Zea-enriched minor LHCs were again investigated on the experimental apparatus used in the reported study. Indeed, the presence of at least one carotenoid radical cation for each minor complex was detected. It is suggested that either the preparation method of incubating the refolded minor LHCs with Zea in contrast to refolding the complexes with only Zea and lutein causes the observed differences or that the observed spectral radical cation signatures are due to experimental artifacts. While the experiments with LHC-II and the minor LHCs gave useful insights into the putative qE mechanism, the quencher site and the mode of action of Zea could still not be unambiguously identified. Most importantly, these studies could not explain the function of the qE keyplayer PsbS. Therefore, the focus of the work was shifted to PsbS protein production, purification and characterisation. In view of inconsistent reports on the pigment binding capacity of this PSII subunit, refolding trials with and without photosynthetic pigments were conducted. The formation of a specific pigmentprotein complex typical for other LHCs was not observed and neither was the earlier reported “activation” of Zea for qE by binding to this protein. Nevertheless, PsbS refolded without pigments displayed secondary structure content in agreement with previous studies, indicating pigment-independent folding. Reconstitution of pigmentfree, refolded PsbS into liposomes confirmed that the protein is stable in the absence of pigments. Zea distributed in PsbS-containing liposomes also showed no spectral alteration that would indicate its “activation”. With the ability to reconstitute PsbS, it was then possible to proceed to modelling qE in a proteoliposome system. For this purpose, PsbS was co-reconstituted with LHC-II, which has been reported to interact with PsbS. One-photon excitation (OPE) and two-photon excitation (TPE) spectroscopy measurements were performed on LHC-II- and LHC-II/PsbS-containing liposomes. This enabled both quantification of Chl fluorescence quenching as well as determination of the extent of electronic Car S1-Chl interactions. The effect of Zea was investigated by incorporating it in the proteoliposome membrane. It was shown that Zea alone was not able to induce significant Chl fluorescence quenching when only LHC-II was present. However, when LHC-II and PsbS were co-reconstituted, pronounced Chl fluorescence quenching and an increase in electronic Car S1-Chl interactions were observed and both effects were enhanced when Zea was present. Western blot analysis indicated the presence of a LHC-II/PsbS-heterodimer in these proteoliposomes. In addition to the OPE and TPE measurements, the average Chl fluorescence lifetime was determined in detergent-free buffer at neutral pH and directly after buffer exchange to low pH. No significant changes in the average lifetime were observed for LHC-II proteoliposomes when either Zea was present or after exchange for low pH buffer. This indicated that Zea alone cannot act as a direct quencher, which concurs with the OPE measurements. Moreover, the complex was also properly reconstituted as no aggregation or significant Chl fluorescence quenching were observed. The average lifetime was not significantly affected in LHC-II/PsbS-proteoliposomes, independent of Zea or pH. However, a shortlived component in the presence of a long-lived component was not resolvable with the time resolution of the fluorescence lifetime apparatus.
Implications for qE model systems and the in vivo quenching mechanism are discussed based on the experiments in detergent solution, on LHC-II aggregates and with the proteoliposome model system.
Die endotheliale NO-Synthase (eNOS) ist im kardiovaskulären System der Hauptproduzent von Stickstoffmonoxid (NO). Studien deuten auf eine Beteiligung der eNOS im Krankheits-verlauf einer Leberzirrhose hin; zirrhotische Tiere zeigen eine reduzierte hepatische eNOS-Aktivität bei unveränderten Proteinmengen. Die reduzierte NO-Menge trägt zu einer Erhöh-ung des intrahepatischen Widerstandes und einer portalen Hypertonie bei. Inhibitoren und posttranslationale Modifikationen der eNOS wurden als auslösende Faktoren postuliert, aber auch am intrazellulären Transport der eNOS beteiligte Proteine könnten eine wichtige Rolle spielen. Ein solches ist das neue Protein NOSTRIN (“eNOS traffic inducer”), das über seine C-terminale SH3-Domäne an eNOS bindet und durch seine N-terminale FCH Domäne an Membranen assoziiert. In vorhergegangenen Studien wurde nur ein translatiertes Protein identifiziert, bezeichnet als NOSTRINalpha. In der vorliegenden Arbeit habe ich eine verkürzte Isoform entdeckt (NOSTRINbeta), die aus einem alternativen Spleißvorgang hervorgeht. Ihr fehlt fast die gesamte FCH Domäne, wodurch keine Membranbindung mehr stattfindet. Diese Isoform konnte nur in pathogenem Lebergewebe nachgewiesen werden. Untersuchun-gen mittels Western Blotting und qRT-PCR mit Proben aus Patienten mit Zirrhose, alkoholischer Hepatitis oder von gesunden Personen, zeigten eine deutliche Erhöhung der Expression beider NOSTRIN-Isoformen von gesundem zu zirrhotischem Gewebe. NOSTRINalpha mRNA-Mengen waren in zirrhotischen vs. gesunden Proben verdoppelt, und in Proben aus Patienten mit zusätzlicher Hepatitis verdreifacht. Dies deutet darauf hin, dass erhöhte Mengen von NOSTRINalpha zu einer Internalisierung und Inaktivierung der eNOS führen könnten. NOSTRINalpha und NOSTRINbeta wurden ebenfalls in Hep3B-Zellen auf Protein und mRNA-Ebene nachgewiesen, ihre Expression war durch Retinsäure zeit- und dosisabhängig stimulierbar. NOSTRINalpha lokalisiert an Plasmamembran und vesikulären Strukturen, NOSTRINbeta hingegen hauptsächlich im Zellkern, eine geringe Fraktion im Zytosol. Über Kartier-ungsstudien wurden zwei nuclear leading sequences (NLS) identifiziert, die den Transport in den Zellkern vermitteln, sowie eine Crm-1-abhängige nuclear export sequence (NES). Im EMSA konnte die Bindung von NOSTRINbeta an die Promotorregion des NOSTRIN-Gens gezeigt werden. Diese Ergebnisse legen eine Funktion von NOSTRINbeta als Transkriptionsfaktor nahe, evtl. innerhalb einer negativen Rückkopplung auf die NOSTRINalpha-Expression. Weiter-führende Studien sollen diesen potentiellen molekularen Mechanismus im Detail klären.
Die primäre Funktion der Haut ist die Abgrenzung des Körpers gegenüber der Umwelt, wodurch der Organismus vor destruktiven Einwirkungen (Xenobiotika, Mikroorganismen, UV-Licht) geschützt wird. Neben wichtigen sensorischen Funktionen ist die Haut außerdem beteiligt an der Regulation von Körpertemperatur sowie Wasserhaushalt und dient als wichtiges Sozialorgan. Da die Verletzung der Haut demnach schwerste Folgen für den Organismus hat, ist die Wundheilung ein essentieller Prozeß zur Aufrechterhaltung der Unversehrtheit der Haut. Der Prozeß der Wund heilung läßt sich in vier Phasen unterteilen (Koagulation, Entzündungsphase, Proliferationsphase, Remodellierungsphase), die zeitlich und räumlich überlappen (Moulin, 1995; Singer and Clark, 1999). ...
Das extrem thermophile Eubakterium Thermus thermophilus ist in den letzten Jahren zu einem Modell für thermophile Organismen geworden und verdankt seinen Statuszum Teil seiner hohe Wachstumsrate, den guten Zellerträgen und der konstitutivenExpression eines natürlichen Kompetenzapparates, der seine genetische Manipulation ermöglicht. Die Verfügbarkeit von kompatiblen Plasmiden und bis zu vier thermostabilen Antibiotikaresistenzmarkern konnten den Wert des Organismus in Hinblick auf biotechnologische Anwendungen noch weiter steigern und tatsächlich besteht nach wie vor ein ungebrochenes Interesse an der Struktur- und Funktionsaufklärung thermophiler Proteine. Der Focus der hier vorliegenden Arbeit richtete sich auf eine der insgesamt zwei terminalen Oxidasen der Atmungskette von T. thermophilus, die Cytochrom ba3 Oxidase. Es wurden verschiedene rekombinante Varianten des Proteins, die sich hinsichtlich der Position und Länge des verwendeten Histidin-Tags unterschieden, kloniert, exprimiert und aufgereinigt. Das Einfügen eines internen His12-Tags in einen periplasmatischen Loop zwischen den Transmembranhelices IV und V führte zu einer rekombinanten Version der Oxidase, die in ihren Eigenschaften dem nativen Wildtyp entsprach und sich durch die in dieser Arbeit etablierte Aufreinigungsstrategie relativ schnell, in guten Ausbeuten und hoher Reinheit aufreinigen ließ. Weiterhin konnten verschiedene Punktmutationen von möglicherweise am Elektronentransfer beteiligten Aminosäureresten generiert und die resultierenden Proteine aufgereinigt und über ihre enzymatische Aktivität charakterisiert werden. Eine weiterführende Charakterisierung der Mutanten erfolgte im Rahmen einer Kooperation und ist bisher noch nicht abgeschlossen. Das Herzstück dieser Arbeit machte jedoch die Definition der Transkriptionseinheit der Cytochrom ba3 Oxidase und die sich daraus ergebenden Fragestellungen aus. So konnte gezeigt werden, dass das ba3 Operon zusätzlich zu den Strukturgenen der dort kodierten Untereinheiten noch mindestens ein, höchst wahrscheinlich jedoch zwei zusätzliche Gene enthält: cbaX und cbaY. Bioinformatische Charakterisierungen ordneten CbaY der diversen Gruppe von sekundären Transportern zu, und es konnte experimentell gezeigt werden, dass seine Anwesenheit für die Expression der Cytochrom ba3 Oxidase förderlich ist. CbaX hingegen konnte über die durchgeführte Homologiesuche keine Funktion zugeordnet werden; uncharakterisierte Homologe waren einzig in der Thermaceae Gruppe zu finden. Durch Deletions- und Komplementationsstudien konnte dem Protein eine entscheidende Rolle in der Assemblierung der ba3 Oxidase bescheinigt werden. CbaX scheint eine zentrale Aufgabe bei der Häm a Insertion in Untereinheit I zu spielen und könnte, ohne Sequenzhomologie aufzuweisen, die Rolle des Surf1-Proteins übernehmen, welches in den sequenzierten Organismen der Thermaceae Gruppe nicht konserviert ist. Die homologe Expression und Aufreinigung von CbaX führte nicht zur erwarteten Ausbeute und Reinheit des Proteins, konnte aber durch immunologische Experimente eine potentielle Interaktion von CbaX und der Cytochrom ba3 Oxidase nachweisen.
The cytochrome bc1 complex or ubiquinol:cytochrome c oxidoreductase (QCR) catalyses electron transfer from ubiquinol to cytochrome c in respiration and photosynthesis coupled to a vectorial proton transport across the membrane, in which the enzyme resides. In both bacteria and eukaryotic organisms, QCR participates in supramolecular assembly of membrane proteins that comprise the respiratory or photosynthetic chain. In the present work, proton transfer pathways, substrate binding and the supramolecular assembly of the respiratory chain in yeast were probed by structure-based site-directed mutagenesis and characterization of the variants. Both active sites centre P, the place of quinol oxidation, and centre N, where quinone reduction takes place, lack direct access to the bulk solvent necessary for proton release and uptake. Based on the X-ray structure, proton transfer pathways were postulated. Analysis at centre P showed, that E272 and Y132 of cytochrome b are important for QCR catalysis as indicated by increased superoxide production and lowered Cyc1p reductase activity in these variants. Pre-steady state heme reduction kinetics in combination with stigmatellin resistance indicated that charge and length of the side chain at position 272 are crucial for efficient docking of the ISP to form the enzyme substrate complex and for electron bifurcation at centre P. Variants of Y312 and F129, both residues of cytochrome b, showed an increased Km indicating participation of these residues in coordination of ubiquinol or the possible intermediate semiquinone anion radical. F129 proved to be crucial for a functional Q-cycle as indicated by respiratory negative growth phenotype and a lowered H+/e- stoichiometry of F129 variants. At centre N, the postulated CL/K and E/R proton transfer pathways are located at opposite sites of the bound ubiquinone. Variants in the surface residues R218 (cytochrome b) and E52 (Qcr7) of the E/R pathway and E82 (Qcr7) of the CL/K pathway showed instability upon purification indicating an important role of these residues for QCR integrity. The slowed down centre N reduction kinetics in H85 (CL/K), R218 and N208 (both E/R) variant was attributed to a destabilised semiquinone anion consistent with the observed decreased sensitivity towards the site-specific inhibitor antimycin and an increased Km. Variants of residues of both pathway, E82Q and R218M, exhibited a decreased H+/e- stoichiometry indicating a crucial role of both residue for maintaining a working Q-cycle and supporting the proposed protonation of the substrate via the Cl/K and the E/R pathway. Long-range interaction between centre N and centre P were observed by altered reduction kinetics of the high potential chain and increased superoxide production in the centre N variants. The role of the cation-pi-interaction between F230 of Cyt1p and R19 of cytochrome c in binding of the redox carrier to QCR was analysed. In F230L hydrophobic interaction were partially lost as was deduced from the ionic strength dependence of Cyc1p reductase activity and Cycp1 binding, as detected by ionic strength sensitive Kd and Km for Cyc1p. The decreased enzymatic rate of F230W could be explained by a disturbed binding of Cyc1p to the variant enzyme. F230 may influence the heme mid point potential and thereby the electron transfer rate to Cyc1p. Reduction of Cobp via both centre P and centre N was disturbed suggesting an interaction between high and low potential chain. Supramolecular association between QCR and cytochrome c oxidase (COX) in yeast mitochondria was probed by affinity chromatography of a his-tagged QCR in the presence of the mild detergent digitonin. In comparison to purification with laurylmaltoside, the presence of both QCR and COX subunits was detected in the elution fractions by SDS-PAGE, Cyc1p reductase and TMPD oxidase activity assays and immunoblot analysis. The CL-dependent formation of the supercomplex between QCR and COX was analysed by replacement variants in the CL-binding site of QCR in CL containing and CL free environment. With an increasing number of replacements of the three lysines the CL-binding pocket supercomplex formation was not abolished, when CL is present as shown by BN-PAGE analysis. This was supported by the synergetic decrease in enzyme activity for both enzymes upon increased number of replacements. In the CL-free environment, no supracomplex formation was observed for a wildtype CL binding site. By replacements of two lysines in the CL-binding pocket, supercomplex formation could be recovered as revealed by BN-PAGE. This indicates, that CL may serve as a charge neutralizer for the lysines near the presumed interaction domain between complex III and complex IV. The obtained results for centre P provide new information of residues critical for stabilisation of ubiquinol and controlling electron short circuit reactions. The observations for centre N variants clearly support the proposed two proton transfer pathways and the role of the bound phospholipids in centre N kinetics. Variants in the Cyc1p binding site suggest a role for F230 both in Cyc1p binding and electron transfer. Clear interaction between the high and low potential chain in both Cyt1p and centre N variants strongly support long-range interactions in the complex. Studies on the supramolecular association of complex III and complex IV indicate a new role of Cl in stabilising a supracomplex.
In dieser Arbeit wurde der Gentransfer von Todesliganden als Ansatz zur Tumor-Gentherapie untersucht. Dazu wurde ein lentiviraler Vektor der zweiten Generationverwendet, der die Todesliganden CD95L oder TRAIL sowie das Markergen EGFP exprimiert. Dies ist die erste Beschreibung von CD95L- oder TRAILexprimierenden lentiviralen Vektoren. Der TRAIL-exprimierende Vektor erwies sich als geeignet für die therapeutische Induktion von Apoptose in humanen Tumorzelllinien auch bei geringen Vektordosen. Die Transduktion mit diesem Vektor bei niedriger MOI führte zu Todesrezeptor-spezifischer Induktion von Apoptose. Diese wurde ausschließlich durch membranständiges TRAIL bei Zell-Zell-Kontakt vermittelt. Die transduzierten Zellen waren zudem in der Lage, bei Kontakt mit nichttransduzierten Zellen in diesen Apoptose auszulösen. Durch den TRAIL-Gentransfer wurde jedoch spezifisch in den transduzierten Zellen Resistenz gegen TRAIL-induzierte Apoptose ausgelöst, während die CD95- oder Cisplatin-induzierte Apoptose nicht beeinflusst war. Dies führte zum Auswachsen einer vollständig TRAIL-resistenten Population. Bereits 72 Stunden nach Transduktion konnten Anzeichen der Resistenz detektiert werden. Der Grund für diese Resistenzinduktion lag in einer spezifischen Blockade der DISC-Bildung und Caspase-8-Aktivierung durch die TRAIL-Todesrezeptoren. Die Signaltransduktion durch den sehr ähnlichen CD95-Signalweg war gänzlich unbeeinflusst. Durchflusszytometrische und proteinbiochemische Untersuchungen der Expression von TRAIL-R1 und TRAIL-R2, sowie die Untersuchung der mRNA-Expression dieser Rezeptoren ergaben, dass beide TRAIL-Todesrezeptoren noch synthetisiert, jedoch intrazellulär zurückgehalten wurden. In fluoreszenzmikroskopischen Versuchen konnte gezeigt werden, dass TRAIL-R2 intrazellulär in einem Komplex mit TRAIL vorlag, der sich im Bereich des ER/Golgi-Apparates befand. Diese Ergebnisse belegen, dass intrazelluläre Interaktion von TRAIL mit seinen Rezeptoren zur Retention dieser Proteine in der Zelle und damit zur Resistenzentwicklung führte. Bei Versuchen zur in vivo-Gentherapie durch lentivirale TRAIL-Expression in humanen Tumortransplantaten auf Nacktmäusen wurde nur ein transienter Effekt erzielt. Es konnte gezeigt werden, dass mit Vektorpartikeln assoziiertes TRAIL-Protein in den Tumoren Apoptose auslöste. Diese Induktion von Apoptose führte zu einer Wachstumsverzögerung. Dadurch wurde jedoch gleichzeitig eine effiziente Transduktion der Tumorzellen mit dem TRAIL-exprimierenden Vektor und ein langfristiger Effekt der TRAIL-Expression verhindert. Diese Ergebnisse zeigen, dass lentiviraler Gentransfer mit konstitutiv TRAIL-exprimierenden Vektoren ungeeignet zur in vivo-Transduktion von Tumoren ist. Gleichzeitig belegen die Ergebnisse der Transduktion mit einem Kontrollkonstrukt einen effizienten Gentransfer. Der hier charakterisierte Resistenzmechanismus ist zuvor nicht beschrieben worden und stellt eine neuartige Form der Therapie-induzierten Resistenz dar. Die proapoptotische Gentherapie durch konstitutive TRAIL-Expression in Tumoren muss nach diesen Ergebnissen neu bewertet werden. Der lentivirale Gentransfer in Tumore in vivo läuft prinzipiell effizient ab. Bei Verwendung eines regulierbaren Expressionssystems und in Kombination mit Suizidgenen könnte eine therapeutische Nutzung des lentiviralen TRAIL-Gentransfers möglich sein.
Survivin wird in einer Vielzahl von Tumoren überexprimiert, während es in normalem Gewebe bis auf einige Ausnahmen kaum detektierbar ist. In den Krebszellen vermittelt Survivin eine erhöhte Resistenz gegenüber der Apoptose-Induktion, was eine Therapie jedoch meist bezweckt. Durch sein differenzielles Expressionsprofil wird Survivin mittlerweile als ein interessanter Angriffspunkt in der Entwicklung einer neuen, zielgerichteten Behandlung von Krebs betrachtet. Aus diesem Grund wurde zu Beginn der vorliegenden Arbeit die Eignung des anti-apoptotischen und Zellzyklus-regulierenden Proteins Survivin als Zielstruktur für eine Krebstherapie im Vergleich zu den veröffentlichten Publikationen verifiziert. Die Analyse der Survivin-Expression in unterschiedlichen Zelllinien ergab, dass sich in Tumorzellen eine charakteristische Überexpression des Survivin-Proteins zeigte im Vergleich zu gesunden, nicht-transformierten Zelllinien. Eine Inhibition der Survivin-Proteinexpression wurde mittels der Methode der RNA-Interferenz erzielt, bei der die Zielzellen mit shRNA-kodierenden Lentiviren infiziert wurden, welche eine gegen die Survivin-mRNA gerichtete Sequenz beinhalteten. Während Survivin-positive Tumorzelllinien und gesunde Endothelzellen eine starke Reduktion in der Lebend-Zellzahl in vitro aufwiesen, waren die Survivin-negativen Kontrollzelllinien von einem Verlust der Survivin-Expression nicht beeinträchtigt. Anschließend erfolgten eine Analyse der Survivin-Abhängigkeit etablierter Tumorzelllinien und die Untersuchung eines Survivin-Verlusts auf die murine Brustdrüsenentwicklung in vivo. Bei einer Inhibition der Survivin-Expression in Krebszellen in einem Transplanationsmodell konnte ein deutlich verzögertes Tumorwachstum beobachtet werden. Dagegen hatte Survivin in der Entwicklung der murinen Brustdrüse keinen Einfluss auf die Rekonstitution des Gewebes und die Proliferation bzw. Differenzierung der Brustepithelzellen. Um einen direkten protein-basierenden Inhibitor des Survivin-Proteins zu entwickeln und das Repertoire an allgemeinen Survivin-Interventionsstrategien zu erweitern, wurde im zweiten Teil der Arbeit mittels des Hefe-Zwei-Hybrid-Systems ein neues Survivin-bindendes Protein isoliert. Nach dem Optimierungsprozess bestehend aus einer Fusion mit einem Trägerprotein zur erleichterten Proteinexpression, der Mutagenese eines Cysteins gegen Serin und der Fusion mit einer Proteintransduktionsdomäne konnte das Protein rekombinant in Bakterien hergestellt und durch Affinitätschromatographie in monomerer Form aufgereinigt werden. Anschließend wurde der Einfluss des artifiziellen, rekombinanten Survivin-inhibierenden Proteins (rSip) auf die Funktionen von Survivin bestimmt. rSip zeigte eine Stabilität von bis zu 14 Stunden im Zellkulturmedium und konnte durch seine C-terminale Proteintransduktionsdomäne in das Zytoplasma der Zielzellen aufgenommen werden. In einer Co-Immunpäzipitation konnte die Bindung von rSip an endogenes Survivin bestätigt werden. In Brustkrebszellen führte rSip in einer Konzentration von 1,5 µM zu einem schnellen Verlust des Survivin-Proteins, was möglicherweise auf eine proteosomale Degradation von Survivin zurückzuführen war. Die Analyse der Konsequenzen einer rSip-Behandlung auf die Funktionen von Survivin in der Apoptose-Inhibition und der Zellzyklus-Progression wurde im letzten Abschnitt der Arbeit durchgeführt. Eine viertägige Inkubation mit 1,5 µM rSip bewirkte eine deutliche Reduktion der Lebend-Zellzahl von bis zu 50% im Falle der Survivin-abhängigen Krebszelllinien. Bei den Survivin-negativen Zelllinien trat dagegen kein veränderter Phänotyp auf. Durch einen TUNEL-Test in Brustkrebszellen konnte gezeigt werden, dass die Ursache für die Abnahme der Zellzahl die Apoptose-Induktion durch rSip ist. In den Zellzyklus-Profilen von rSip-behandelten Krebszellen konnte ebenfalls ein starker Anstieg in der apoptotischen Zell-Population beobachtet werden. Abschließend lässt sich sagen, dass in der vorliegenden Arbeit neben der Methode der lentiviralen Applikation von Survivin-spezifischen shRNA-Sequenzen eine neue Möglichkeit der Interferenz mit der Survivin-Funktion in Krebszellen vorgestellt wurde. Die Entwicklung des Survivin-inhibierenden Proteins rSip steht zugegebenermaßen erst am Anfang. Die ersten hier präsentierten Ergebnisse zeigen jedoch klar ein Potential dieses vielversprechenden direkten Survivin-Inhibitors als ergänzende Wirkstoffklasse auf dem Gebiet der therapeutischen Proteine zu den bereits existierenden niedermolekularen Substanzen bzw. antisense-Oligonukleotiden, die auf Ebene der Transkription bzw. der Translation von Survivin wirken.
The transcription factor p63 is part of the p53 protein family, which consists of three members, p53, p63 and p73. P63 shares structural similarity with all family members, but is associated to different biological functions than p53 or p73. While p53 is mainly linked to tumor suppression and p73 is connected with neuronal development, p63 has been connected to critical biological roles within ectodermal development and skin stem cell biology as well as supervision of the genetic stability of oocytes. Due to its gene structure p63 is expressed as at least six different isoforms, three of them containing a N-terminal transactivation domain. The isoforms that are of biological relevance both have a C-terminal inhibitory domain that negatively regulates the transcriptional activity. This inhibitory domain is supposed to contain two individual components of which one is internally binding and masking the transactivation domain while the other one can be sumoylated. To further investigate this domain a mutational analysis with the help of transactivation assays in SAOS2 cells was carried out to identify the critical amino acids within the inhibitory domain and the impact on transcriptional activity of TAp63alpha, the p63-isoform which is essential for the integrity of the female germline. The results of these experiments show that a stretch of approximately 13 amino acids seems to be important for the regulation of transcriptional activity in TAp63alpha, due to the increased transcriptional activity occurring in this region after mutation. Additional experiments showed that this mechanism is distinct from sumoylation, which seems to have only implications for the intracellular level of TAp63alpha. As a conclusion, the C-terminus of the Tap63alpha is essential for two different mechanisms, which control the transcriptional activity of the protein. Both regulatory elements are independent from each other and can now be restricted to certain amino acids. Activation of the wild type protein might take place in the identified region via post-translational modification. Furthermore an inhibition assay was carried out to test if the same region might have implications on the second biological relevant isoform deltaNp63alpha. The results show that the same amino acids which show an impact on transcriptional activity in Tap63alpha lead to a significant change in functional behaviour of deltaNp63alpha. There is a possibility that both proteins are regulated with opposite effects via the same mechanisms, based at the C-terminus of the p63alpha-isoforms. In both cases a modification of these residues could lead to a more opened conformation of the protein with consequences on promoter binding, which can be even important for deltaNp63alpha with respect to promoter squelching. Both alpha-isoforms seem to be regulated via the C-terminus and to elucidate if that is also the case for TAp63gamma a deletion analysis was carried out. The results show that there are also amino acids within the C-terminus of TAp63gamma, which have implications on the transcriptional activity of the protein. Therefore the C-terminus seems to play a major role for regulation of diverse p63 isoforms.
My graduate thesis is on the "Structural studies of membrane transport proteins". Transporters are membrane proteins that have multiple membrane-spanning a-helices. They are dynamic and diverse proteins, undergoing a large conformational change and transporting wide range of susbtrates. Based on their energy source they can be classified into primary and secondary transport systems. Primary transport systems are driven by the use of chemical (ATP) or light energy, while secondary transporters utilize ion gradients to transport substrates. I began my PhD dissertation on secondary transporters by two-dimensional crystallization and electron crystallographic analysis and recently my focus also has shifted towards 3D crystallization. The following projects constitute my PhD thesis: 1) 2D crystallization of MjNhaP1 and pH induced structural change: MjNhaP1, a Na+/H+ antiporter that is regulated by pH has been implicated in homeostasis of H+ and Na+ in Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85°C. MjNhaP1 was cloned and expressed in E. coli. Two-dimensional crystals were obtained from purified protein at pH4. Electron cryo-microscopy yielded an 8Å projection map. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4-6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1was studied in situ in 2D crystals revealing a major change in density within the helix bundle relative to the dimer interface. This change occurred at pH6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter, respectively. This is the first instance where a conformational change associated with the regulation of a secondary transporter appears to map structurally. Reconstruction of 3D map and high-resolution structure by x-ray crystallography would be necessary to understand the mechanism of ion transport and regulation by pH. 2) 2D crystallization of Proline transporter: Proline transporter (PutP) from E.coli belongs the sodium-solute symporter family that includes disease related sodium dependent glucose and iodide transporter in humans. Sodium and proline are co-transported with a stoichiometry of 1:1. Purified PutP was reconstituted to yield 2D crystals that were hexagonal in nature. The 2D crystals had tendency to stack indicating their willingness to form 3D crystals. A projection map of PutP from negatively stained crystals showed trimeric arrangement of protein. Other members of the SSF family have been shown to be monomers. My analysis of oligomeric state of PutP in detergent by blue native gel indicates a monomer in detergent solution. It is likely that PutP can function as a monomer but at higher concentration and in lipid bilayer it tends to form trimer. 3) Oligomeric state and crystallization of carnitine transporter from E.coli: E.coli carnitine transporter (CaiT) belongs to the BCCT (Betaine, Carnitine and Choline) superfamily that transports molecules with quaternary amine groups. CaiT is predicted to span the membrane 12 times and acts as a L-carnitine/g-butyrobetaine exchanger. Unlike other members in this transporter family, it does not require an ion gradient and does not respond to osmotic stress. Over-expression of the protein yielded ~2mg of protein/L of culture. The structure and oligomeric state of the protein were analyzed in detergent and lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. Gel filtration and cross-linking studies further support this. Reconstitution of CaiT into lipid bilayers resulted in 2D crystals. Analysis of negatively stained 2D crystals confirmed that CaiT is a trimer in the membrane. Initial 3D crystallization trials have been successful and currently, the crystals diffract to 6Å and are being improved. 4) Monomeric porin OmpG: OmpG is a bacterial outer membrane b-barrel protein. It is monomeric and its size (33kDa) places it as a prime candidate for a structural solution, using the recently developed method of solid state NMR (work in collaboration with Prof.Hartmut Oskinat, FMP, Berlin). A long-term aim would be to study porins as templates for designing nanopores, for DNA sequencing and identification. I have expressed OmpG in inclusion bodies and refolded at an efficiency of >90% into a functional form using detergent. OmpG was then crystallized by 2D crystallization yielding an 8Å projection map whose structure was similar to native protein. In addition, these crystals were used for structure determination by solid state NMR. An initial spectrum of heavy isotopically labeled OmpG has allowed identification of specific amino acid residues including threonine and proline. Additionally, I obtained 3D crystals in detergent that diffract to 5.5Å and are being improved.
Three-dimensional structure of the glycine-betaine transporter BetP by cryo electron crystallography
(2008)
The soil bacterium Corynebacterium glutamicum has five secondary transporters for compatible solutes allowing it to cope with osmotic stress. The most abundant of them, the transporter BetP, performs a high affinity uptake of glycine-betain when encountering hyperosmotic stress. BetP belongs to the betaine/carnitine/choline/transporter (BCCT) family, and is predicted to have twelve transmembrane helices with both termini facing the cytoplasm. The goal of this thesis is to facilitate understanding of BetP function by determining a three dimensional (3D) model of its structure. Two-dimensional (2D) crystallization of wild-type (WT) BetP has been successfully performed by reconstitution into a mixture of E. coli lipids and bovine cardiolipin, which resulted in vesicular crystals diffracting to 7.5 Å resolution (Ziegler, Morbach et al. 2004). Diffraction patterns of these crystals however showed unfocused spots, generally due to high mosaicity. Better results were obtained by using the constitutively active mutant BetPdeltaC45 in which the first 45 amino acids of the positively charged C-terminus were removed. BetPdeltaC45 crystals obtained under the same conditions for BetP WT were concluded to be pseudo crystals, based on the inconsistence of symmetry. These crystals had BetPdeltaC45 molecules randomly up/downwards inserted into membrane crystals, and cannot be used for structure determination, even though they diffracted up to 7 Å. The problem of pseudo crystal formation could be solved by changing the lipids used for 2D crystallization to a native lipid extract from C. glutamicum cells. This change of lipids improved the crystals to well-ordered packing with exclusive p121_b symmetry. To understand the role of lipids in crystal packing and order, lipids were extracted at different stages during crystallization, and identified by using multiple precursor ion scanning mass spectrometry. The results show that phosphatidyl glycerol (PG) 16:0-18:1 is the most dominant lipid species in C. glutamicum membranes, and that BetP has a preference for the fatty acid moieties 16:0-18:1. Crystallization with synthetic PG 16:0-18:1 proved that an excess of this lipid prevents pseudo crystal formation, but these crystals did not reach the quality as previously achieved by using the C. glutamicum lipids. Apart from the effect of lipids in crystallinity, the concentration and type of salts influenced crystal growth and morphology. High salt conditions (>400 mM LiCl or KCl) yielded tubular crystals, whereas low salt conditions (<300 mM LiCl, NaCl or KCl) led to formation of up to 10 µm large sheet-like crystals. The intermediate concentration gave a mixture of sheet-like and tubular crystals. In terms of resolution, sheets diffracted better than tubes. The sheet-like crystals used for 3D map reconstruction were obtained from a dialysis buffer containing 200 mM NaCl combined with using C. glutamicum lipids. Electron microscopic images were taken from frozen-hydrated crystals using a helium-cooled JEOL 300 SFF microscope or a liquid nitrogen-cooled FEI Tecnai G2 microscope at 300 kV, which allowed optimal data collection and minimized radiation damage to the sample. More than 1000 images of tilt angles up to 50° were taken and evaluated using optical diffraction of a laser beam. The best 200 images were processed with the MRC image processing software package, and 79 images from different tilt angles were merged to the final data set used for calculation of a 3D map at a planar resolution of 8 Å. The structure shows BetPdeltaC45 as a trimer with each monomer consisting of 12 transmembrane alpha-helices. Protein termini and loop regions could not be determined due to the limited resolution of the map. Six of the twelve helices line a central cavity forming a potential substrate-binding chamber. Each monomer shows a central cavity in different sizes and shapes. Thus, the constitutively active BetPdeltaC45 thus forms an unusual asymmetric homotrimer. BetP most likely reflects three different conformational states of secondary transporters: the cytoplasmically open (C), the occluded (O), and the periplasmically open (P) states. The C and O states are similar to BetP WT projection structure, while the P state is discrepant and highly flexible due to the shape and size of the central cavity as well as the lowest intensity of the density. The observation of the P state corresponds well to the constitutively active property of BetPdeltaC45. For the high resolution structure of the C and O states are available, this work presents the first structural information of the P state of a secondary transporter.
Membrane proteins play vital role in a variety of cellular processes, such as signal transduction, transport and recognition. In turn they are involved in numerous human diseases and currently represent one of the most prevalent drug targets. A comprehensive understanding of the mechanisms mediated by membrane proteins requires information about their structures at near-atomic resolution, although structural studies of membrane proteins remain behind those of soluble proteins. A bottleneck in the study of membrane proteins resides in the difficulties that are encountered during their high-level production in cell based systems. However, many toxic effects attributed to the over production of membrane proteins are eliminated by cell-free expression, as viable host cells are no longer required. Therefore, the objective of this study was to obtain adequate amounts of selected membrane transport proteins for their structural studies using a cell-free expression system. For the establishment of the cell-free system for membrane proteins, the transporters YbgR and YiiP from Salmonella typhimurium LT2, PF0558 and PF1373 from Pyrococcus furiosus, from the cation diffusion family (CDF), BetP from Corynebacterium glutamicum from the betaine/carnitine/choline transporter (BCCT) family and Aq-2030 from Aquifex aeolicus VF5 from the monovalent cation/proton antiporter-2 (CPA2) family were selected. An Escherichia coli S-30 extract based cellfree system was established by generating the best expression constructs of the target proteins, preparing T7 RNA polymerase and an S-30 extract with high translation efficiency. The functionality of the S-30 extract was shown by the cell-free expression of correctly folded Green Fluorescent Protein (GFP). Essential factors of the cell-free system such as the Mg2+ concentration, the bacterial S-30 extract proportion in the reaction mixture and the time-course of cell-free reactions have been optimized. For the cell-free production of membrane proteins in soluble form, the possibility to supplement cell-free reactions with detergents was explored. A wide range of non-ionic or zwitterionic detergents, were found to be compatible with cell-free synthesis, while ionic detergents and non-ionic detergents at high concentrations had an inhibitory effect. Moreover, high concentrations of polyoxyethylene-alkyl-ethers (Brij) detergents were found to have enhancing effect on the production levels as well as on the solubility of cell-free produced proteins. As membrane proteins tend to misfold and aggregate in a membrane-free translation system, the possibility to supplement the cell-free reactions with inner membrane vesicles (IMVs) to obtain correctly folded target transport proteins was explored. All the target proteins were successfully produced in the batch cell-free reactions and were found to be incorporated in the IMVs. A continuous exchange cell-free (CECF) system was established, where consumable substrates (amino acids, nucleotides and energy regenerating compounds) were supplied to the cell-free reaction mixture through a dialysis membrane, which in consequence resulted in high-level production of target proteins compared to the batch system. The osmosensing and osmoregulated sodium-coupled symporter BetP from C. glutamicum was chosen for the large scale production in CECF set-up. The protein is easily produced in E. coli and is functional as assayed by its transport activity, after purification and reconstitution in liposomes. It is therefore possible to compare in-vivo and cell-free production. High-level cell-free production of BetP was achieved in CECF mode in different forms: (i) as precipitate, (ii) as soluble form in detergent, and (iii) incorporated in IMVs. Cell-free production of BetP resulted in the yield of about 0.5 mg of purified BetP from 1 ml of CECF reaction. The yield of purified BetP was increased to 1.6 fold by addition of 1% polyoxyethylene-(20)-cetyl-ether (Brij58) detergent in the reaction mixture. Moreover, the high level cell-free production of BetP (0.5 mg purified BetP/ml reaction mixture) incorporated in IMVs was shown for the first time in this work.However, it was observed that oligomerization of BetP was not efficient in the cell-free system. Factors that can promote the folding of membrane proteins such as lipids and chaperones were investigated. Addition of lipids and molecular chaperone GroE facilitated correct folding of BetP resulting in increased yield and stability of cell-free produced BetP. The results obtained indicate that most of the cell-free produced BetP exists in functional oligomeric form. The possibility of obtaining milligram amounts of BetP, a 12 trans-membrane protein from the cell-free reactions holds promise for structural and functional studies of other membrane proteins. In any case, the strategies adapted in this study should prove extremely valuable for the production of membrane proteins in the E. coli cell-free expression system.
In der vorliegenden Arbeit wurden die Porcinen Endogenen Retroviren (PERV) erstmals ausführlich biochemisch und biologisch charakterisiert sowie ihr Wirtsspektrum und ihre Expression in vitro näher analysiert. Es wurden sensitive immunologische Nachweismethoden entwickelt, die in experimentellen, präklinischen und ersten klinischen Studien zur Xenotransplantation angewandt wurden. Die Charakterisierung der Viren ergab, daß es sich um TypCRetroviren handelt, wobei ein Teil der Partikel nicht gereift ist. In hoch gereinigten Viruspräparationen aus Überständen produzierender porciner und PERVinfizierter, humaner Zellen konnten alle Strukturproteine identifiziert werden. Dazu wurden neben Elektrophoresen Western BlotAnalysen eingesetzt, wobei kreuzreaktive Antiseren gegen verwandte Viren und neu entwickelte, erstmals gegen PERVProteine gerichtete Antiseren benutzt wurden. Bei der Untersuchung der biologischen Eigenschaften wurde gezeigt, daß PERV in vitro ein mit anderen Retroviren vergleichbares immunsuppressives Potential besitzt. Mit diesen neuen Antiseren und den hoch gereinigten Viruspräparationen wurden sehr sensitive Western BlotVerfahren sowie ELISAs auf der Basis synthetischer Peptide als Nachweissysteme etabliert und validiert. Zuerst wurden Seren gesunder Blutspender mit diesen Tests untersucht. Dann wurden Seren von Schlachtern und von Patienten nach Xenotransplantationen getestet. Erstmals wurden auch Seren von Pavianen, denen u.a. auch ganze Schweineorganen transplantiert wurden und die eine profunde, für zukünftige Anwendungen typische, pharmakologische Immunsuppression erhielten, auf Antikörper gegen PERV untersucht. Bei Seren einiger Patienten und gesunder Blutspendern wurden zwar Kreuzreaktionen mit viralen Proteinen gefunden, es lagen aber keinerlei Anzeichen für eine PERVInfektion vor. Um das Wirtspektrum von PERV zu analysieren, wurden Zellinien und erstmals auch primäre Zellen verschiedener Spezies einschließlich Ratte und Maus mit PERV inokuliert. Dabei wurden erstmals Hinweise auf die Suszeptibilität von humanen Blutzellen erhalten. Es konnte gezeigt werden, daß mitogenstimulierte porcine PBMC PERV freisetzen können, wobei alle drei Subtypen der Hüllproteine nachgewiesen wurden. Eine nähere Analyse ergab daß verschiedene Schweinerassen aber auch einzelne Individuen der gleichen Rasse unterschiedliche Mengen an Virus freisetzen. Unter bestimmten Stimulationsbedingungen wurde PERV von adhärenten Zellen produziert, die nicht näher charakterisiert werden konnten. Eine endgültige Beurteilung des Infektionsrisikos bei Xenotransplantationen ist nach Abschluß dieser Studie noch nicht möglich. Mit der Charakterisierung von PERV und den immunologischen Nachweismethoden wurden die Voraussetzung für die Analyse weiterer experimenteller und klinischer Xenotransplantationen gelegt.
Isolierung und Charakterisierung der Atmungsketten-Superkomplexe aus Paracoccus denitrificans
(2004)
Isolierung von Atmungsketten-Superkomplexen aus Paracoccus denitrificans: Im Rahmen der vorliegenden Arbeit wurde ein Reinigungsprotokoll zur Präparation der Atmungskettenkomplexe I, III und IV des Gram-negativen, fakultativ anaeroben Bodenbakteriums Paracoccus denitrificans in Form eines NADH Oxidase Komplexes erstellt. Bisher konnten stabile Superkomplexe bakterielle Atmungskettenkomplexe nur in Gestalt einer Chinol Oxidase, bestehend aus den Komplexen III (Ubichinol:Cytochrom c Oxidoreduktase) und IV (Cytochrom c Oxidase), isoliert werden (Berry, et al., 1985). Jedoch enthielten diese Assemblierungen keinen Komplex I (NADH: Ubichinon Oxidoreduktase). Dies ist die erste chromatographische Isolierung eines kompletten "Respirasoms" aus Paracoccus denitrificans. Unter der Verwendung des milden Detergenz Digitonin zur Solubilisierung von Paracoccus denitrificans Membranen gefolgt von zwei chromatographischen Reinigungsschritten, der Hydroxylapatit-Chromatographie und der Gelfiltration, konnte eine NADH Oxidase, bestehend aus den Komplexen I, III und IV in einer 1:4:4 Stöchiometrie isoliert werden. Neben der Isolierung der NADH Oxidase konnten weitere kleine Superkomplexe identifiziert werden, die aus vier Komplex III und vier Komplex IV (III4IV4) sowie vier Komplex III und zwei Komplex IV (III4IV2) assoziiert waren. Charakterisierung der Superkomplexe aus Paracoccus denitrificans: Die isolierten Atmungskettenkomplexe wurden zur Charakterisierung bezüglich ihrer Funktion, enzymatischen Aktivität, Stöchiometrie und Untereinheiten-Zusammensetzung aus Paracoccus denitrificans Wildtyp-Membranen analysiert. Proben aller Präparationsstufen wurden parallel zur BN-Gelelektrophorese und für enzymatische Einzel- und kombinierte Aktivitäten der Komplexe I-IV eingesetzt. Mittels BN-Gelelektrophorese konnten die apparenten Massen der Komplexe bestimmt werden. Im folgenden denaturierenden SDS-Gel wurde die Untereinheiten-Zusammensetzung der Superkomplexe durch Immunodetektion mit Antikörpern gegen die Komplexe I, III, IV und Cytochrom c552 analysiert. Abschließend konnte nach der Gelfiltration festgestellt werden, das Komplex II eindeutig nicht Teil des Superkomplexes (I1III4IV4) war. Die Stöchiometrie des Superkomplexes a wurde mit Hilfe der fluorimetrischen Bestimmung von FMN (Flavin) als Marker für Komplex I und mittels Pyridin Hämochromogen Spektren für Häm a, b und c bestimmt. Da Komplex III physiologisch als Dimer vorliegt (Mayer et al. 2002), müsste die Stöchiometrie der funktionellen Einheit des Superkomplex a folgendermaßen lauten: I1 (III2)2 IV4. In diesem Fall diente der Vergleich der Wechselzahl einzelner Präparationsstufen als Maß der Verunreinigung der ergab, dass Fremdkomplexe mit Flavin und Cytochrom b im Ausgangsmaterial der Präparation vorhanden waren. Nur die Wechselzahl des Komplexes IV blieb während der Präparation konstant. Um den Gehalt an Komplex I und die Qualität der Präparation abzuschätzen, wurde das Verhältnis der HAR zu dNADH:DBQ Oxidoreduktase Aktivität in Membranen und isoliertem Superkomplex a bestimmt. Es war während der Präparation konstant. Die Bestimmung des Phospholipidgehalts aus isoliertem Superkomplex a im Vergleich zu P. denitrificans Membranen ergab eine Abnahme von 980 ± 80 nmol PL / mg Protein in Membranen auf 290 ± 10 nmol / mg Protein in isoliertem Superkomplex a, wohingegen der Ubichinongehalt von 2,7 ± 0,3 nmol / mg auf 6,7 ± 0,8 nmol / mg in isolierter Oxidase anstieg. Katalytische Aktivitäten von P. denitrificans Membranen des Parentalstamms und verschiedener Mutantenstämme zeigten, dass die Inaktivierung des Gens für fest gebundenes Cytochrom c552 die Bildung eines Superkomplexes nicht verhindern konnte, was ein Hinweis dafür ist, dass dieses Elektronen Carrier Protein nicht essentiell für die strukturelle Verbindung zwischen den Komplexen III und KIV ist. Komplex I Aktivität wurde ebenfalls in Membranen von Mutanten-Stämmen, denen Komplex III oder Komplex IV fehlte, gefunden. Jedoch enthielten diese Stämme keinen assemblierten Komplex I, sondern nur dissoziierte Untereinheiten des Komplexes. Trotz der Verwendung der selben Protokolle für die elektrophoretische Trennung und chromatographische Isolierung wie für Superkomplexe aus dem Wildtyp-Stamm, führte die Isolierung aus Mutantenstämmen, denen Komplex III oder IV fehlte, zum vollständigen Verlust der NADH:DBQ Oxidoreduktaseaktivität. Dies weist darauf hin, dass Paracoccus denitrificans Komplex I durch die Assemblierung in Form eines NADH Oxidase Superkomplex stabilisiert wird. Zusätzlich zum Substrat Channeling scheint die strukturelle Stabilisierung von Membran Protein Komplexen die Hauptaufgabe von respiratorischen Superkomplexen zu sein.